Method of screening remedy for renal failure

ABSTRACT

A screening tool and a convenient screening method for obtaining a renal failure treating agent, and a pharmaceutical composition for treating renal failure and a production method thereof are disclosed. The aforementioned screening tool is a G protein coupling type receptor FGK which is a polypeptide capable of activating CTGF promoter, a functionally equivalent modified substance thereof or a homologous polypeptide, or a cell expressing the aforementioned polypeptide. The screening method is a method which employs inhibition of the aforementioned polypeptide as the index.

TECHNICAL FIELD

This invention relates to a cell expressing orphan GPCR and a method forscreening an agent for treating renal failure which uses thereof.

BACKGROUND OF THE INVENTION

Chronic dialysis patients due to renal diseases are increasing everyyear. Since this is a serious problem in terms of medical economy too,great concern has been directed toward the improvement of its progress.The causes of renal disorders are various such as chronic glomerularnephritis, diabetic nephropathy and hypertensive nephrosclerosis, andorigins thereof also are various such as an immunological mechanism andhypertension. However, irrespectively the cause, a common developingmechanism is considered in glomerular disorders at a certain stage. Thisis a point of view based on the excess filtration (hyperfiltration)theory proposed by Brenner et al. (Non-patent Reference 1). Outline ofthe hyperfiltration theory is described in the following, that is, astate of so-called glomerular hypertension is induced by the increase ofglomerular pressure caused by a change in the hemodynamics in the kidneydue to functional nephron reduction, hypertension, hyperglycemia,excessive protein ingestion and the like resulting from glomerulusdisorders. This induces a glomerular cell disorder. As a result, itaccelerates character conversion of mesangial cells and production ofextracellular matrices and further develops into glomerulosclerosis.When functional nephron is reduced by this, glomerular pressure of theremaining functional nephron is further increased. The hypertrophy ofremnant glomeruli occurs to compensate the function-lost nephrons in thebeginning, but finally causes physical failure. The glomerulosclerosisis acceleratively progressed by this vicious circle and results in thelast phase renal failure. This glomerulosclerosis and the productionacceleration of extracellular matrices in the renal tubulointerstialcell are generally called renal fibrosis which is a histological changethat coincides with the progress of renal failure which is irrespectiveof the original disease (Non-patent Reference 2). Accordingly, althoughit is needless to say that the treatment of individual original diseaseis important, it is considered that inhibition of the fibrosis as afinal common pathway is an effective therapeutic approach.

It is known that various cytokines and growth factors are concerned inthe fibrosis, and it is considered that TGF-β among them is the mostimportant progressing factor since, for example, it induces productionof extracellular matrix constituting proteins such as fibronectin andtype I collagen(Non-patent Reference 3) and inhibits expression/functionof enzymes degrading the extracellular matrix (Non-patent Reference 4).In addition, inhibition of the increase of extracellular matrices of thekidney by the expression/function inhibition of TGFβ using a TGFβneutralizing antibody (Non-patent Reference 5), an antisenseoligonucleotide (Non-patent Reference 6) decholin (Non-patent Reference7) or a TGFβ receptor neutralizing antibody (Non-patent Reference 8) hasbeen shown by nephritic animal models and its effectiveness has alreadybeen revealed. Thus, it has been shown that the inhibition of TGF-βsignal leads to the renal failure treatment based on inhibition of renalfibrosis as the mechanism. However, since TGFβ is an important cytokinewhich has anti-inflammatory action and tumor growth inhibiting activityin addition to the fibrosis accelerating activity, and TGFβ knockoutmice die of autoimmune disease (Non-patent Reference 9), it wasconsidered that for the inhibition of fibrosis, inhibition of a factorwhich mediates fibrosis acceleration activity of TGFβ at its downstreamis more desirable than direct inhibition of TGFβ (Non-patent Reference10).

In recent year, a new cytokine connective tissue growth factor(CTGF)whose expression is induced by TGFβ was discovered (Non-patent Reference11), and it has been reported that production of an extracellular matrixis induced by CTGF expression (Non-patent Reference 12), that expressionof collagen induced by TGFβ is inhibited by a CTGF antisenseoligonucleotide and an anti-CTGF antibody (Non-patent Reference 13),that expression of CTGF is increased in a human renal fibrosispathologic tissue image (Non-patent Reference 14), that expression ofCTGF is increased together with TGFβ in a rat pathological modelkidney(Non-patent Reference 15), and that hepatocyte growth factorinhibits fibrosis of a mouse pathological model kidney via inhibition ofCTGF production (Non-patent Reference 16). When these were generalized,a mechanism in which TGFβ induces expression of CTGF in the renaltissue, and CTGF further accelerates production of extracellularmatrices together with TGF-β, thereby accelerating the fibrosis, hasbeen revealed (Non-patent Reference 17). That is, it has been revealedthat CTGF is a cytokine positioned at the downstream of TGFβ in therenal fibrosis, showing that it can become a new therapeutic target(Non-patent Reference 18). In addition, relation to pathological stateshas been pointed out such as that type I collagen whose expression isinduced by TGFβ and CTGF shows low expression in human normal kidney andits expression increased in the glomerulus and tubular epithelium ofpathological state kidney (Non-patent Reference 19), and that expressionof type I collagen is increased by the induction of intrinsic TGFβexpression by high glucose in cultured mouse mesangial cells (Non-patentReference 20). On the other hand, CTGF is expressed in broad range oftissues such as brain, placenta, lung, liver, kidney, skeletal muscleand the like, and its expression has no tissue specificity (Non-patentReference 22).

FGK (fibrogenic GPCR in kidney) is an orphan GPCR identical to the GPR91reported in 2001 (Non-patent Reference 21) Human GPR91 and mouse GPR91are polypeptides respectively consisting of 330 and 317 amino acids, andit is considered that they are seven times transmembrane type receptorshaving 7 transmembrane regions. The human and mouse GPR91 moleculesmutually have a homology of about 68%. Expression of the human GPR91 isfound only in the kidney, and expression of the mouse GPR91 is slightlyfound in the liver in addition to the kidney. Although it has beenreported that UTP functions as a ligand of human GPR91 in a reactionsystem which uses Xenopus egg (Patent Reference 1), its G proteincoupling with GPR91 and physiological function remain unknown. Althoughsequences having homology with FGK are disclosed in Patent References 2to 9, there is only description of information on expression other thanthat they are adenosine receptors (Patent Reference 9) and a P2U2purinergic receptors (Patent Reference 8). Thus, their physiologicalfunctions are not known.

(Patent Reference 1)

International Publication No. 97/20045 pamphlet

(Patent Reference 2)

International Publication No. 97/24929 pamphlet

(Patent Reference 3)

International Publication No. 01/98351 pamphlet

(Patent Reference 4)

International Publication No. 00/22131 pamphlet

(Patent Reference 5)

International Publication No. 01/90304 pamphlet

(Patent Reference 6)

International Publication No. 00/31258 pamphlet

(Patent Reference 7)

International Publication No. 02/00719 pamphlet

(Patent Reference 8)

International Publication No. 02/61087 pamphlet

(Patent Reference 9)

U.S. Ser. No. 02/137,887

(Non-patent Reference 1)

The New England Journal of Medicine, (USA), 1982, vol.307, pp.652-659

(Non-patent Reference 2)

Journal of the American Society of Nephrology, (USA), 1996, vol.7,pp.2495-2508

(Non-patent Reference 3)

The Journal of Biological Chemistry, (USA), 1987, vol.262, pp.6443-6446

(Non-patent Reference 4)

Journal of the American Society of Nephrology, (USA), 1999, vol.10,pp.790-795

(Non-patent Reference 5)

Nature, (England), 1990, vol.346, pp.371-374

(Non-patent Reference 6)

Kidney International, (USA), 1996, vol.50, pp.148-155

(Non-patent Reference 7)

Nature, (England), 1992, vol.360, pp.361-364

(Non-patent Reference 8)

Kidney International, (USA), 2001, vol.60, pp.1745-1755

(Non-patent Reference 9)

Nature, (England), 1992, vol.359, pp.693-699

(Non-patent Reference 10)

Kidney International, (USA), 1997, vol.51, pp.1388-1396

(Non-patent Reference 11)

Molecular Biology of the Cell, (USA), 1993, vol.4, pp.637-645

(Non-patent Reference 12)

The Journal of Investigative Dermatology, (USA), 1996, vol.107,pp.404-411

(Non-patent Reference 13)

The FASEB Journal, (USA), 1999, vol.13, pp.1774-1786

(Non-patent Reference 14)

Kidney International, (USA), 1998, vol.53, pp.853-861

(Non-patent Reference 15)

American Journal of Physiology Renal Physiology, (USA), 2002, vol.282,pp.F933-F942

(Non-patent Reference 16)

The FASEB Journal, (USA), 2003, vol.17, pp.268-270

(Non-patent Reference 17)

Journal of the American Society of Nephrology, (USA), 2001, vol.12,pp.472-484

(Non-patent Reference 18)

Kidney International, (USA), 2000, vol.58, pp.1389-1399

(Non-patent Reference 19)

Kidney International, (USA), 2002, vol.62, pp.137-146

(Non-patent Reference 20)

The Journal of Clinical Investigation, (USA), 1994, vol.93, pp.536-542

(Non-patent Reference 21)

Journal of Molecular Biology, (England), 2001, vol.307, pp.799-813

(Non-patent Reference 22)

Circulation, (USA), 1997, vol.95, pp.831-839

DISCLOSURE OF THE INVENTION

As a result of intensive studies, the inventors of the present inventionhave found that FGK which is an orphan GPCR is expressed inkidney-specifically and activates the promoter of CTGF which is atherapeutic target of renal failure. Based on the knowledge, a methodfor screening a substance capable of inhibiting CTGF expression usingFGK inhibition as a marker, namely a method for screening an agent fortreating renal failure based on the CTGF expression inhibition byselecting a FGK inhibitor, was constructed. In addition, it was foundthat FGK, even by itself alone, can be used as a screening tool for anagent for treating renal failure using a change of its activity, andfound that an inverse agonist of FGK surely inhibits expression of CTGF.Based on this, a method for screening an agent for treating renalfailure by selecting an FGK inverse agonist was established. Asdescribed in the foregoing, since CTGF is expressed in a broad range oftissues and its expression has no tissue specificity, when a screeningis carried out simply using the promoter region of CTGF gene, theresulting substance inhibits production of CTGF not only in the kidneybut also in all tissues, so that there is a danger of causingside-effects based on the inhibition of cell growth and extracellularmatrix production as the actions of CTGF. On the other hand, when asystem for screening a substance capable of inhibiting CTGF expressionusing FGK inhibition as a marker which is constructed based on theknowledge found by the inventors of the present invention that FGKactivates the CTGF promoter is used, since FGK is kidney-specificallyexpressed, it is expected that the inhibition of CTGF production by aresulting substance of the screening is kidney-specific and itsexpression in other tissues does not occur.

As a result of these, the inventors of the present invention haveaccomplished the invention by providing a convenient method forscreening an agent for treating renal failure, and a pharmaceuticalcomposition for treating renal failure and a production method thereof.

That is, the invention relates to,

(1) A screening tool for an agent for treating renal failure, which is apolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2, or a polypeptide comprising an amino acid sequence represented bySEQ ID NO:2 in which from 1 to 10 amino acids are deleted, substitutedand/or inserted and which is capable of activating CTGF promoter.

(2) The screening tool for an agent for treating renal failure, which isa cell expressing the polypeptide described in (1).

(3) A method for detecting whether or not a test compound is an inverseagonist, which comprises

a step of allowing the cell described in (2) co-expressing a chimeric Gprotein in which C-terminal amino acid sequence is the amino acidsequence represented by SEQ ID NO:16, and which is a chimera of apartial polypeptide having promoting activity of phospholipase Cactivity of a phospholipase C activity-promoting G protein with apartial polypeptide having a Gi receptor coupling activity, to contactwith a test compound, and

a step of analyzing a change in activity of the polypeptide described in(1) in said cell.

(4) A method for screening an agent for treating renal failure, whichcomprises

a step of allowing the cell described in (2) co-expressing a chimericG-protein in which C-terminal amino acid sequence is the amino acidsequence represented by SEQ ID NO:16, and which is a chimera of apartial polypeptide having promoting activity of phospholipase Cactivity of a phospholipase C activity-promoting G protein with apartial polypeptide having a Gi receptor coupling activity, to contactwith a test compound, and

a step of analyzing a change in activity of the polypeptide described in(1) in said cell.

(.5) A method for screening a substance inhibiting expression of CTGF,which comprises

a step of allowing the cell described in (2) expressing the DNA of SEQID NO:13 having a reporter gene in downstream to contact with a testcompound, and

a step of measuring the reporter activity in said cell.

(6) The screening method according to (5), wherein the substanceinhibiting expression of CTGF is an agent for treating renal failure.

(7) A method for screening an agent for treating renal failure, whichcomprises

a step of allowing the cell described in (2) expressing the DNA of SEQID NO:14 having a reporter gene in downstream to contact with a testcompound, and

a step of measuring the reporter activity in said cell.

(8) A pharmaceutical composition for treating renal failure, whichcomprises an inverse agonist for the polypeptide described in (1).

(9) A pharmaceutical composition for treating renal failure, whichcomprises a substance obtainable by the method according to one of (4)to (7).

(10) A method for producing a pharmaceutical composition for treatingrenal failure, which comprises

a step of screening using the method according to one of (4) to (7), and

a step of preparing a pharmaceutical composition using a substanceobtained by the screening.

(11) A method for treating renal failure, which comprises administeringan effective amount of an inverse agonist for the polypeptide described(1) and/or a substance obtaiable by the method according to one of (4)to (7) to a subject in need of the treatment of renal failure.

(12) Use of an inverse agonist for the polypeptide described in (1)and/or a substance obtainable by the method according to one of (4) to(7) for the manufacture of a pharmaceutical composition for treatingrenal failure.

Patent Reference 1 describes a receptor having the same sequence of FGKwhich is a polypeptide as the screening tool of the present invention.It is described that this is expressed in the kidney and activated byATP, ADP, UTP and UDP and that this is useful as a tool for screening.However, there is no description regarding its usefulness as a tool forwhat object of the screening, and its illustrative use. Patent Reference2 to Patent Reference 9 disclose molecules having homology with FGK.Patent Reference 6 shows that the molecule is expressed in the kidney,but there is no description regarding its illustrative use. Although inPatent Reference 7, renal disease and renal failure are included invarious diseases cited to which the molecule relates, there is nodescription supporting the same. Patent Reference 8 discloses a largenumber of GPCR including molecules having high homology with FGK, and alarge number of diseases in which a large number of GPCR are concerned.Although renal disease is included therein, their illustrative examplesare not described and there are no experimental supports on their use.Patent Reference 9 discloses a receptor having homology with FGK anddiscloses a method for identifying agonists or antagonists thereof. Itis described that these agonists and antagonists are useful for treatingvasodilatation, hypotension, chronic renal disease, thyroid disease andimmune diseases including asthma. However, it only describes that themolecule is expressed in specific tissues including the kidney as thebasis that they are useful for treating chronic renal disease, and doesnot describes the relationship between the molecule and CTGF.Relationship between a molecule having homology with FGK and renaldisease and relationship between the molecule and CTGF are not describedin any of Patent References 2 to 5.

Accordingly, the fact that FGK activates the promoter of CTGF which is atherapeutic target of renal failure is novel knowledge found by theinventors of the present invention, and the method for screening asubstance capable of inhibiting CTGF expression using FGK inhibition asa marker, the method for screening an agent for treating renal failureby selecting a FGK inverse agonist, and the pharmaceutical compositionfor treating renal failure and the production method thereof areinventions provided for the first time by the inventors of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing luciferase activity when pCTGF-luc orpCOLIA2-luc reporter plasmid was transfected into HEK293 cells, andchanges in the activity caused by FGK gene cotransfection. Ordinate ofthe graph shows relative value of the luciferase activity.

FIG. 2 is a graph showing luciferase activity when pEF-BOS-Gqi andpSRE-luc reporter plasmids were cotransfected into HEK293 cells, andchanges in the activity caused by FGK gene transfection. Ordinate of thegraph shows relative value of the luciferase activity.

BEST MODE FOR CARRYING OUT THE INVENTION

The following describes the invention in detail.

Firstly, the terms used in the present invention are described.

The “FGK” as used herein means “FGK protein”, and the “inverse agonist”represents a substance which inhibits spontaneous activity (namely anactivity detected by activated FGK existing in a equilibrium state inthe absence of a FGK ligand or agonist) of a polypeptide for screeningof the invention (e.g., FGK). The method for judging whether or not apolypeptide “activates the CTGF promoter” is not particularly limited,but it can be judged for example by verifying whether or not thepolypeptide shows a dose-dependent transactivation under the conditionsdescribed in Example 3. The “Gi” is one of the subfamily of G proteinwhich functions as a signal transduction amplifying factor into cells bycoupling with a receptor, and is a G protein that inhibits the activityof adenylate cyclase. When the adenylate cyclase activity is inhibited,for example, concentration of intracellular cAMP is reduced. The“phospholipase C activity-promoting G protein” is one of the subfamilyof G protein which functions as a signal transduction amplifying factorinto cells by coupling with a receptor, and is a G protein whichpromotes the activity of phospholipase C. When the activity ofphospholipase C is promoted, for example, intracellular Ca²⁺concentration increases. As the phospholipase C activity promoting Gprotein, for example, Gq can be cited.

<Screening Tool for an Agent for Treating Renal Failure>

A screening tool of the present invention for an agent for treatingrenal failure includes a polypeptide type screening tool for an agentfor treating renal failure and a cell type screening tool for an agenttreating renal failure. The “screening tool” as used herein means asubstance to be used for the screening (illustratively a polypeptide ora cell expressing the polypeptide, which is used in the screening). The“screening tool for an agent treating renal failure” is a polypeptide orcell as the subject to be contacted with a test compound in the methodfor screening an agent for treating renal failure of the presentinvention, for screening an agent for treating renal failure. Use of theaforementioned polypeptide described in [1] or cell described in [2] forthe screening of an agent for treating renal failure is also included inthe present invention.

(1) Polypeptide type screening tool for an agent for treating renalfailure

The polypeptide type screening tool for an agent for treating renalfailure includes

1) the screening tool for an agent for treating renal failure which is apolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2;

2) the screening tool for an agent for treating renal failure which is apolypeptide that comprises an amino acid sequence in which from 1 to 10amino acids of the amino acid sequence represented by SEQ ID NO:2 aredeleted, substituted and/or inserted and which can activate CTGFpromoter (to be referred to as functionally equivalent modifiedsubstance hereinafter); and

3) the screening tool for an agent for treating renal failure which is apolypeptide that comprises an amino acid sequence having 90% or more ofhomology with the amino acid sequence represented by SEQ ID NO:2 andwhich can activate CTGF promoter (to be referred to as homologouspolypeptide hereinafter).

Among the polypeptide type screening tools for an agent for treatingrenal failure, the polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO:2 is a member of the commonly known humanorphan GPCR, and the polypeptide is called human FGK.

As the functionally equivalent modified substance which can be used as apolypeptide type screening tool for an agent for treating renal failure,“a polypeptide which comprises an amino acid sequence in which from 1 to10, preferably from 1 to 7, more preferably from 1 to 5, amino acids ofthe amino acid sequence represented by SEQ ID NO:2 are deleted,substituted and/or inserted and which can activate CTGF promoter” ispreferable.

Among the homologous polypeptides, a protein which comprises an aminoacid sequence having a homology of preferably 90% or more, morepreferably 95% or more, further preferably 98% or more, regarding theamino acid sequence represented by SEQ ID NO:2 is preferable, and morepreferably which can activate CTGF promoter. In this connection, theaforementioned “homology” as used herein means a value (Identities)obtained by Clustal program (Higgins and Sharp, Gene, vol. 73, pp.237-244, 1998; Thompson et al., Nucleic Acid Res., vol. 22, pp.4673-4680, 1994) retrieval using parameters prepared by default. Theaforementioned parameters are as follows. As Pairwise Aliment ParametersK tuple 1 Gap Penalty 3 Window 5 Diagonals Saved 5

Various polypeptides which can be used as the polypeptide type screeningtool for an agent for treating renal failure, namely human FGK,functionally equivalent modified substances and homologous proteins, arehereinafter called polypeptides for a screening tool.

Not only mutants in human but also FGK or mutants thereof derived fromorganisms other than human (e.g., mouse, rat, hamster or dog) areincluded in the polypeptides for a screening tool. Proteins artificiallymodified by genetic engineering techniques based on these naturalproteins (that is, human derived mutants, or FGK derived from organismsother than human or mutants thereof) or human FGK are also included. Inthis connection, the “mutant” (variation) as used herein means anindividual difference found in the same protein within the same species,or a difference found in homologous protein among several species.

The mutants of human FGK in human, or FGK derived from organisms otherthan human or mutants thereof, can be obtained by those skilled in theart based on the information on a nucleotide sequence of human FGK gene(e.g., the nucleotide sequence represented by SEQ ID NO:1). In thisconnection, unless otherwise noted, the genetic engineering techniquescan be carried out in accordance with the conventionally known methods(Maniatis, T. et al., “Molecular Cloning—A Laboratory Manual”, ColdSpring Harbor Laboratory, NY, 1982, and the like).

For example, a desired protein can be obtained by designing appropriateprimers or probe based on the information on the nucleotide sequence ofhuman FGK gene, carrying out PCR or hybridization using theaforementioned primers or probe and a sample (e.g., a total RNA or mRNAfraction, a cDNA library or a phage library) derived from a organism ofinterest [e.g., a mammal (e.g., human, mouse, rat, hamster or dog)],thereby obtaining a gene of the protein, expressing the gene using anappropriate expression system, and then confirming that the thusexpressed protein can activate the CTGF promoter by, for example, themethod described in Example 3.

In addition, regarding the aforementioned proteins artificially modifiedby genetic engineering techniques, a desired protein can be obtained byobtaining a gene of the protein by a usual method, for example,site-specific mutagenesis (Mark, D. F. et al., Proc. Natl. Acad. Sci.USA, 81, 5662-5666, 1984), expressing the gene using an appropriateexpression system, and then confirming that the thus expressed proteincan activate the CTGF promoter by, for example, the method described inExample 3.

The polypeptide for a screening tool can be obtained by variousconventionally known methods, for example, it can be prepared byconventionally known genetic engineering techniques using a gene codingfor the protein of interest. More illustratively, it can be prepared byculturing a cell or transformed cell (that is, a transformed cell whichis transfected with an expression vector containing a DNA coding for thepolypeptide for a screening tool and expressing the aforementionedpolypeptide) which is described later, under such a condition that thepolypeptide for a screening tool can be expressed, and then separatingand purifying the protein of interest from the culture mixture bymethods generally used for the separation and purification of receptorproteins.

In preparing a polypeptide for a screening tool, the method forobtaining a gene encoding the same is not particularly limited, but forexample, in case that human FGK is prepared, a DNA consisting of thenucleotide sequence represented by SEQ ID NO:1 can for example be usedas the gene coding for the same. In this connection, the codonscorresponding to a desired amino acid are well known by themselves, andtheir selection may be optional and, for example, they can be determinedin accordance with a usual method by taking codon usage of the host tobe used into consideration (Crantham, R. et al., Nucleic Acids Res., 9,r43-r74, 1981).

The DNA consisting of the nucleotide sequence represented by SEQ ID NO:1can be obtained, for example, by ligating DNA fragments produced by achemical synthesis method, or can be obtained by the polymerase chainreaction (PCR) method (Saiki, R. K. et al., Science, 239, 487-491, 1988)using a cDNA library derived from a cell or tissue having the ability toproduce human FGK as the template and using an appropriate primer setdesigned based on the nucleotide sequence represented by SEQ ID NO:1. Asthe aforementioned cell or tissue having the ability to produce humanFGK, for example, human kidney and the like can be cited. Also, as theaforementioned primer set includes such as a combination of anoligonucleotide consisting of the nucleotide sequence represented by SEQID NO:3 and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO:4.

Although the separation and purification methods which can be used inthe preparation of a polypeptide for a screening tool are notparticularly limited, they can be carried out, for example, by thefollowing procedures. For example, a cell membrane fraction containing apolypeptide for a screening tool can be obtained by culturing a cellexpressing the polypeptide for a screening tool on its surface,suspending them in a buffer, and then homogenizing to centrifuge them.The polypeptide for a screening tool can be purified by solubilizing thethus obtained cell membrane fraction, and then carrying out itstreatment with a general protein precipitating agent, ultrafiltration,various types of liquid chromatography [e.g., molecular sievechromatography (gel filtration), adsorption chromatography, ion exchangechromatography, affinity chromatography, high performance liquidchromatography (HPLC) and the like], dialysis, or a combination thereof.In this connection, characteristics of the receptor can be maintainedeven after the solubilization by the use of a solubilizing agent as mildas possible (e.g., CHAPS, Triton X-100, digitonin or the like) insolubilizing the cell membrane fraction.

When the polypeptide for the screening tool is prepared, confirmation ofexpression of the aforementioned polypeptide, confirmation of itsintracellular localization, its purification or the like can be easilycarried out by inframe-fusing a polypeptide for a screening tool with anappropriate marker sequence and expressing it, if necessary. Theaforementioned marker sequence includes FLAG epitope, hexa-histidinetag, hemagglutinin tag and myc epitope. In addition, by insertion of aspecific sequence recognized by a protease (e.g., enterokinase, factorXa, thrombin or the like) between the marker sequence and polypeptidefor a screening tool, digestion and removal of the marker sequence partby such a protease is possible. For example, there is a report statingthat muscarine acetylcholine receptor and hexa-histidine tag wereconnected with a thrombin recognizing sequence (Hayashi, M. K. and Haga,T. J. Biochem., 120, 1232-1238, 1996).

(2) Cell type screening tool for an agent for treating renal failure

The cell type screening tool of the present invention for an agent fortreating renal failure includes

1) a screening tool for an agent for treating renal failure which is acell expressing human FGK;

2) a screening tool for an agent for treating renal failure which is acell expressing a functionally equivalent modified substance; and

3) a screening tool for an agent for treating renal failure which is acell expressing a homologous protein.

The cell which can be used as the cell type screening tool of thepresent invention for an agent for treating renal failure (to bereferred to as cell for screening tool hereinafter) is not particularlylimited, as far as it expresses the aforementioned polypeptide for ascreening tool when used as a cell type screening tool for an agent fortreating renal failure, and it can be a transformed cell in which theaforementioned polypeptide is artificially expressed, or it can be anatural cell or a cell strain thereof which is known to express apolypeptide for a screening tool. However, a transformed cell in whichthe aforementioned polypeptide is artificially expressed is preferable.

The host cell which can be used for preparing various types oftransformed cell which can be used as the cell type screening tool ofthe present invention for an agent for treating renal failure(to bereferred to as transformed cell for screening tool hereinafter) is notparticularly limited, as far as it expresses a polypeptide for ascreening tool, and its examples include generally used conventionallyknown microorganisms such as Escherichia coli or a yeast (Saccharomycescerevisiae), or conventionally known cultured cells such as a vertebratecell (e.g., CHO cell, HEK293 cell or COS cell) or an insect cell (e.g.,Sf9 cell).

The aforementioned vertebrate cell includes such as COS cell as a monkeycell (Gluzman, Y., Cell, 23, 175-182, 1981), a dihydrofolate reductasedeficient strain of Chinese hamster ovary cell (CHO) (Urlaub, G andChasin, L. A., Proc. Natl. Acad. Sci. USA, 77, 4216-4220, 1980), a humanfetal kidney-derived HEK293 cell, or a 293-EBNA cell (Invitrogen) inwhich Epstein-Barr virus EBNA-1 gene is transferred into theaforementioned HEK293 cell.

The expression vector which can be used for preparing a transformed cellfor a screening tool is not particularly limited, with as far as it canexpress a polypeptide for a screening tool, and it can be optionallyselected in accordance with the kind of a host cell to be used.

For example, as the expression vector for vertebrate cells, those whichhave a promoter, a RNA spice site, a polyadenylation site, atranscription termination sequence and the like locating at the upstreamof a gene to be expressed can generally be used, and they may furtherhave a replication origin, if necessary. Example of the aforementionedexpression vector includes such as pSV2dhfr having SV40 early promoter(Subramani, S. et al., Mol. Cell. Biol., 1, 854-864, 1981), pEF-BOShaving a human elongation factor promoter (Mizushima, S. and Nagata, S.,Nucleic Acids Res., 18, 5322, 1990) and pCEP4 having cytomegaloviruspromoter (Invitrogen).

More illustratively, when COS cell is used as the host cell, anexpression vector which has the SV40 replication origin, can performautonomous replication in COS cell, and further has a transcriptionpromoter, a transcription termination signal and an RNA splice site canbe used, and examples thereof include pME18S (Maruyama, K. and Takebe,Y., Med. Immunol., 20, 27-32, 1990), pEF-BOS (Mizushima, S. and Nagata,S., Nucleic Acids Res., 18, 5322, 1990) and pCDM8 (Seed, B., Nature,329, 840-842, 1987).

The aforementioned expression vector can be incorporated into COS cell,by such as DEAE-dextran method (Luthman, H. and Magnusson, G., NucleicAcids Res., 11, 1295-1308, 1983), calcium phosphate-DNA co-precipitationmethod (Graham, F. L. and van der Ed, A. J., Virology, 52, 456-457,1973), a method which uses a cationic liposome reagent (Lipofectamine;Gibco BRL), electroporation (Neumann, E. et al., EMBO J., 1, 841-845,1982) or the like.

Also, when CHO cell is used as the host cell, a transformed cell whichstably produces a polypeptide for a screening tool can be obtained bycotransfection of an expression vector containing a DNA coding for thepolypeptide for a screening tool together with a vector capable ofexpressing a neo gene which functions as a G418 resistance marker, suchas pRSVneo (Sambrook, J. et al, “Molecular Cloning—A Laboratory Manual”,Cold Spring Harbor Laboratory, NY, 1989), or pSV2-neo (Southern, P. J.and Berg, P., J. Mol. Appl. Genet., 1, 327-341, 1982), and thenselecting a G418-resistant colony.

In addition, when 293-EBNA cell is used as the host cell, pCEP4(Invitrogen) or the like which has the replication origin ofEpstein-Barr virus and can perform autonomous replication in 293-EBNAcell can be used as the expression vector.

The transformed cell for a screening tool can be cultured in accordancewith the usual method, and a polypeptide for a screening tool isproduced in the cell or on the cell surface. As the medium which can beused in the aforementioned culture, generally used various media can beoptionally selected in accordance with the employed host cell. Forexample, in the case of COS cell, a medium such as Dulbecco's ModifiedEagle's minimum essential medium (DMEM) or the like further supplementedwith fetal bovine serum (FBS) or the like serum component as occasiondemands can be used. Also, in the case of 293-EBNA cell, Dulbecco'sModified Eagle's minimum essential medium (DMEM) or the like mediumsupplemented with fetal bovine serum (FBS) or the like serum componentcan be used by further adding G418-thereto.

The transformed cell for a screening tool is not particularly limited sofar as it expresses a polypeptide for a screening tool. It is desirablethat the transformed cell for a screening tool expresses a G protein inwhich its C-terminal amino acid sequence is the amino acid sequencerepresented by SEQ ID NO;16 (Asp-Cys-Gly-Leu-Phe), in addition to thepolypeptide for a screening tool. The amino acid sequence represented bySEQ ID NO;16 is an amino acid sequence consisting of the C-terminal 5amino acid residues of Gi, and the “G protein in which its C-terminalamino acid sequence is the amino acid sequence represented by SEQ IDNO;16” is called “C terminal Gi type G protein” in the following.

The aforementioned C terminal Gi type G protein include such as (1) Gior (2) a chimeric G protein in which its C-terminal amino acid sequenceis the amino acid sequence represented by SEQ ID NO:16, and which is achimera of a partial polypeptide having promoting activity ofphospholipase C activity of a phospholipase C activity-promoting Gprotein (e.g., Gq) with a partial polypeptide having a Gi receptorcoupling activity. In the following, the chimeric G protein of a partialpolypeptide having a phospholipase C activity promoting activity of Gqwith a partial polypeptide having a Gi receptor coupling activity iscalled Gqi.

The polypeptide for a screening tool binds to Gi by recognizing an aminoacid sequence consisting of the C-terminal 5 amino acid residues of Gi(namely, the amino acid sequence represented by SEQ ID NO;16).Accordingly, the polypeptide for a screening tool can binds to not onlyGi but also Gqi. When the polypeptide for a screening tool and Cterminal Gi type G protein are expressed in the transformed cell for ascreening tool, these polypeptides can be bonded together inside thecell.

The “partial polypeptide having promoting activity of phospholipase Cactivity of Gq” according to the aforementioned Gqi is not particularlylimited, as far as that it does not contain the C terminal amino acidsequence and it has the promoting activity of phospholipase C activity.An example thereof includes an N terminal side partial polypeptide of Gqin which the amino acid sequence consisting 5 amino acid residues inC-terminal is deleted.

The “partial polypeptide having a Gi receptor conjugating activity”according to the aforementioned Gqi is not particularly limited, with asfar as it contains the amino acid sequence consisting of 5 amino acidresidues in the C-terminal of Gi and it does not have the activity toinhibit activity of adenylate cyclase. An example thereof includes a Cterminal side partial polypeptide of Gi consisting of the amino acidsequence represented by SEQ ID NO:16.

<Inverse Agonist Detection Method>

Using the aforementioned polypeptide for a screening tool or cell for ascreening tool as a detection tool, whether or not a test compound is aninverse agonist of the polypeptide for screening of the presentinvention (preferably FGK) can be detected. A polypeptide for screeningwhich activates the promoter of CTGF as a target of an agent fortreating renal failure (e.g., FGK) can be used as a screening tool foran agent for treating renal failure even by itself alone. An inverseagonist of the polypeptide for screening (preferably FGK) is an usefulsubstance as an agent for treating renal failure. According to thedetection method of the present invention, detection of a change of theactivity of a polypeptide for screening (e.g., FGK) is carried out bymeasuring an index of activity according to a physiologicalcharacteristic of the protein to be used for the screening. The index isfor example a change in the Ca²⁺ concentration or a change in the amountof cAMP. Illustratively, the detection methods described in thefollowing can be exemplified. As the polypeptide for screening, a cellexpressing the receptor, a membrane fraction of the cell, a purifiedpreparation of the protein or the like can also be used.

The method of the present invention for detecting whether or not a testcompound is an inverse agonist of the polypeptide for screening(preferably FGK) include

1) a method for detecting whether or not it is an inverse agonist of thepolypeptide for screening (preferably FGK) using a change ofintracellular Ca²⁺ concentration as an index (that is, a Ca²⁺ typedetection method);

2) a method for detecting whether or not it is an inverse agonist of thepolypeptide for screening (preferably FGK) using a change of the amountof intracellular cAMP as an index (that is, a cAMP type detectionmethod); and

3) a method for detecting whether or not it is an inverse agonist of thepolypeptide for screening. (preferably FGK) using of a GTPγS bindingmethod (to be referred to as GTPγS binding type detection methodhereinafter). These detection methods are explaind in order. 1) Ca²⁺type detection method The Ca²⁺ type detection method of the inventionuses a cell co-expressing (i) a polypeptide for a screening tool and(ii) a chimeric G protein (e.g., Gqi) in which its C-terminal amino acidsequence is the amino acid sequence represented by SEQ ID NO:16 andwhich is a chimera of a partial polypeptide having promoting activity ofphospholipase C activity of a phospholipase C activity-promoting Gprotein with a partial polypeptide having a Gi receptor couplingactivity (to be referred to as cell for Ca²⁺ type detectionhereinafter). When whether or not it is an inverse agonist is detectedby the Ca²⁺ type detection method of the present invention, the cell forCa²⁺ type detection is allowed to contact with a test compound, and achange in the Ca²⁺ concentration inside the aforementioned cell for Ca²⁺type detection is directly or indirectly analyzed (namely, measured ordetected). Regarding the change in Ca²⁺ concentration, a change in theCa²⁺ concentration can be directly analyzed using, for example, acalcium binding fluorescence reagent (e.g., fura2 or fluo3 or the like),or a change in the Ca²⁺ concentration can be indirectly analyzed byanalyzing the transcriptional activity of a gene in which itstranscriptional level is controlled depending on the Ca²⁺ concentration[e.g., a gene in which an activator protein 1 (AP1) responding sequenceis inserted into upstream of a luciferase gene].

When that the cell for Ca²⁺ type detection is allowed to contact with atest compound, if Ca²⁺ concentration in the cell for Ca²⁺ type detectionis reduced, it can be judged that it is an inverse agonist of thepolypeptide for screening (preferably FGK). In this connection, it isdesirable to carry out the same operation using a control cell in whichthe polypeptide for a screening tool is not expressed but Gqi isexpressed, or the host cell before transformation as a control insteadof the cell for Ca²⁺ type detection co-expressing the polypeptide for ascreening tool, and Gqi and thereby to confirm that the intracellularCa²⁺ concentration of the aforementioned control cell or theaforementioned host cell is not reduced.

More illustratively, this can be carried out making use of the method ofExample 4 or Example 5.

As described above, Gi is not directly used but Gqi is used as thecoupling protein in the Ca²⁺ type detection method of the presentinvention, so that the detection of whether or not a sample is aninverse agonist can be carried out by analyzing not the cAMPconcentration but the Ca²⁺ concentration. In general, the Ca²⁺concentration can be measured more conveniently and quickly incomparison with the cAMP concentration.

2) cAMP Type Detection Method

In the cAMP type detection method of the present invention, a cell for ascreening tool is used as the cell for cAMP type detection. Since Gi isconstitutively expressed in general host cells, a cell for cAMP typedetection can be obtained by using a natural cell or a cell strainthereof which is known to express a polypeptide for a screening tool, orby transforming a host cell with an expression vector containing a DNAcoding for a polypeptide for a screening tool.

When whether or not it is an inverse agonist is detected by the cAMPtype detection method of the present invention, the cell for cAMP typedetection is allowed to contact with a test compound, and a change inthe cAMP concentration inside the aforementioned cell for cAMP typedetection is directly or indirectly analyzed (namely, measured ordetected). With regard to the change in cAMP concentration, a change inthe cAMP concentration can be directly analyzed using, for example, acommercially available cAMP measuring kit (Amersham or the like), or achange in the cAMP concentration can be indirectly analyzed by analyzingthe transcription activity of a gene in which its transcriptional levelis controlled depending on the cAMP concentration [e.g., a gene in whicha cAMP responsive element (CRE) is inserted into upstream of aluciferase gene].

In case that the cell for cAMP type detection is allowed to contact witha test compound, when cAMP concentration inside the cell for cAMP typedetection is increased, it can be judged that the aforementioned testcompound is an inverse agonist for FGK. Also, it is desirable to carryout the same operation using a cell in which the polypeptide for ascreening tool is not expressed as a control, instead of the cell forcAMP type detection expressing the polypeptide for a screening tool andGi, and thereby to confirm that the cAMP concentration inside theaforementioned cell is not increased by the aforementioned testcompound. More illustratively, for example, this can be carried out bydetecting whether or not the cAMP concentration is increased when a testcompound is contacted, in comparison with a case when the test compoundis not contacted when a low dose (e.g., 0.2 μg) of FGK is expressedunder the same condition of Example 4.

3) GTPγS Binding type Detection Method

By the GTPγS binding type detection method of the present invention,whether or not a sample is an inverse agonist for FGK can be detected,using the GTPγS binding method (Lazareno, S. and Birdsall, N. J. M., Br.J. Pharmacol., 109, 1120-1127, 1993) in which a polypeptide for ascreening tool, a cell membrane fraction containing the aforementionedpolypeptide or a cell expressing the aforementioned polypeptide areused. A cell or cell membrane fraction for GTPγS binding type detectionmethod (a cell for GTPγS type detection) can be obtained by using anatural cell or a cell strain thereof which is known to express apolypeptide for a screening tool, or by transforming a host cell with anexpression vector containing a DNA coding for a polypeptide for ascreening tool.

For example, this can be carried out by the following procedure.

That is, a cell membrane containing a polypeptide for a screening toolis mixed with ³⁵S-labeled GTPγS (400 pmol/L) in a mixed solution of 20mmol/L HEPES (pH 7.4), 100 mmol/L NaCl, 10 mmol/L MgCl₂ and 50 mmol/LGDP. After incubation in the presence or absence of a test compound, thereaction mixture is filtered through a glass filter or the like, and theradioactivity of GTPγS remained on the filter is measured using a liquidscintillation counter or the like. Whether or not the sample is aninverse agonist for FGK can be detected by using reduction of thespecific GTPγS binding in the presence of the test compound as an index.Also, it is desirable to carry out the same operation using a cellmembrane in which the polypeptide for a screening tool is not expressedas a control, instead of the cell membrane expressing the polypeptidefor a screening tool, and thereby to confirm that the GTPγS binding isnot reduced in the presence of the aforementioned test compound.

<Method for Screening a Substance which Inhibits CTGF expression>

A method for screening a substance which inhibits CTGF expression, usinginhibition of activity of a polypeptide for screening (preferably FGK)as an index is included in the present invention. A reporter assaysystem which uses the promoter region of a CTGF gene can be used in themethod. As already described above, it has been revealed that CTGF is acytokine locating downstream of TGFβ in renal fibrosis, showing thatCTGF is shown to be a target for therapeutic for an agent for treatingrenal failure. The inventors of the present invention have found thatFGK activates the CTGF promoter considered to be the target for a drugcreation for renal failure as shown in Example 3 which is describedlater, and further established a method for screening a substance whichinhibits CTGF expression, using activity inhibition of FGK activity asan index.

The method of the present invention for screening a substance whichinhibits CTGF expression with the feature comprising

a step of allowing a cell for a screening tool expressing the DNA (CTGFpromoter) of SEQ ID NO:13having a reporter gene in its downstream tocontact with a test compound, and

a step of measuring the reporter activity in the aforementioned cell,and a substance which inhibits expression of CTGF can be screened.

As the cell to be used in the method, a cell coexpressing (i) apolypeptide for a screening tool and (ii) a reporter gene fused with aCTGF promoter region (to be referred to as a cell for CTGF promoter typedetection hereinafter) can be used. In this connection, with regard tothe aforementioned cell, it is desirable that the host cell beforesubjecting to the transformation is a cell derived from the kidney. Assuch cell, for example, the aforementioned HEK293 cell can be cited.

The reporter gene assay (Tamura et al., Method for StudyingTranscription Factors (written in Japanese), published by Yodo-sha,1993) is a method for detecting expression regulation of genes usingexpression of a reporter gene as the marker. Expression regulation of agene is generally controlled at a site called promoter region existingin its upstream of 5′ end, and the quantity of gene expression at thetranscription stage can be estimated by measuring the activity of thispromoter. When a test substance activates the promoter, it activatestranscription of a reporter gene located in the downstream of thepromoter region. In this manner, the promoter activation activity,namely the expression acceleration activity, can be detected byreplacing it with the expression of the reporter gene. Accordingly, theactivity of a test compound on the expression regulation of CTGF can bedetected by the reporter gene assay using the CTGF promoter region, byreplacing it with the expression of the reporter gene. The “reportergene” fused with the CTGF promoter region consisting of the nucleotidesequence represented by SEQ ID NO:16 is not particularly limited so longas it is a generally used one, but an enzyme gene or the like which canbe quantitatively easily measured is desirable. For example, a bacterialtransposon-derived chloramphenicol acetyltransferase gene (CAT), afirefly-derived luciferase gene (Luc) a jellyfish-derived greenfluorescent protein gene (GFP) and the like can be cited. The reportergene should be functionally fused with the promoter region of CTGFconsisting of the nucleotide sequence represented by SEQ ID NO:16. Testcompound-dependent changes in the induction of transactivation can beanalyzed by comparing expression quantities of the reporter gene whenthe cell for CTGF promoter type detection is allowed to contact and notallowed to contact with a test compound.

It is desirable to carry out the same operation using a cell for ascreening tool in which the polypeptide for a screening tool is notexpressed but the reporter gene fused with the CTGF promoter region isexpressed as a control, instead of the cell for CTGF promoter typedetection and thereby to confirm that the reporter activity in theaforementioned cell for a screening tool is not inhibited by theaforementioned test compound.

By carrying out the aforementioned process and selecting a substancewhich inhibits the reporter activity, screening of a substance capableof inhibiting expression of CTGF can be carried out. Illustratively, theaforementioned screening can be carried out by the method described inExample 3. For example, by adding a sets compound under the assaycondition described in Example 3, a substance having an IC₅₀ value of 10μM or less, preferably a substance having an IC₅₀ value of 1 μM or less,more preferably a substance having an IC₅₀ value of 0.1 μM or less,under the condition of Example 3 can be selected as a substance havingthe activity to inhibit CTGF expression. More preferably, this can becarried out in the same manner under the condition of Example 7.

<Method for Screening an Agent for Treating Renal Failure>

An agent for treating renal failure can be screened using the screeningtool for an agent for treating when the renal failure of the presentinvention (includes both of the polypeptide type screening tool for anagent for treating renal failure and cell type screening tool for anagent for treating renal failure).

As already described, it has been revealed that CTGF is a cytokinelocating downstream of TGFβ in renal fibrosis, showing that CTGF isshown to be a target for therapeutic for an agent for treating renalfailure, namely a substance which inhibits expression of CTGF is shownto be an agent for treating renal failure. Also, it has been revealedthat FGK is localized in the kidney. Additionally, the inventors of thepresent invention have found that FGK activates the CTGF promoter andalso activates the human type I collagen alpha two subunit (COLIA2)promoter as described in Example 3. According to these findings, aninverse agonist of a polypeptide for screening (preferably FGK) or asubstance which inhibits expression of CTGF by inhibiting FGK activityis useful as an agent for treating renal failure. Thus, the polypeptidefor a screening tool itself or cell for a screening tool itself so fardescribed can be used as a screening tool for the screening of an agentfor treating renal failure.

The test compounds which can be subjected to screening using a screeningtool for an agent for treating renal failure of the present inventionare not particularly limited. For example, various conventionally knowncompounds (including peptides) registered in the chemical file, a groupof compounds obtained by combinatorial chemistry techniques (Terrett,.N. K. et al., Tetrahedron, 51, 8135-8137, 1995) or a group of randompeptides prepared by applying the phage display method (Felici, F. etal., J. Mol. Biol., 222, 301-310, 1991) and the like can be used. Also,microbial culture supernatants, natural components of plants or marineorganisms, or animal tissue extracts and the like can be used as testcompounds of the screening. Additionally, compounds (including peptides)obtained by chemically or biologically modifying the compounds(including peptides) selected by the screening tool for an agent fortreating renal failure of the present invention can be used.

The screening method of the present invention is roughly divided intothe following three types based on the respective detection methods, anda substance useful as an agent for treating renal failure can bescreened using any one of these method or a combination thereof. Thescreening methods of the present invention:

(1) a screening method which uses activity change of a polypeptide forscreening (preferably FGK) as the index, (2) a method for screening asubstance which inhibits CTGF expression, using activity inhibition of apolypeptide for screening (preferably FGK) as the index, and

(3) a screening method which uses activity change of a polypeptide forscreening (preferably FGK) as the index, using the COLIA2 promoter aredescribed below in order.

In the (1) a screening method which uses activity change of apolypeptide for screening (preferably FGK) as the index,

1) a method for screening an inverse agonist using a change ofintracellular Ca²⁺ concentration as the index (to be referred to as Ca²⁺type screening method hereinafter);

2) a method for screening an inverse agonist using a change ofintracellular cAMP quantity as the index (to be referred to as cAMP typescreening method hereinafter); and

3) a method for screening an inverse agonist using the GTPγS bindingassay (to be referred to as GTPγS binding type screening methodhereinafter) are included and these are described in order in thefollowing.

1) Ca²⁺ Type Screening Method

The Ca²⁺ type screening method of the present invention is notparticularly limited, so far as it includes a step for detecting whetheror not it is an inverse agonist by the Ca²⁺ type detection method of thepresent invention and a step for selecting an inverse agonist.

The inverse agonist can be screened by the aforementioned Ca²⁺ typedetection method using reduction of Ca²⁺ concentration in the cell forCa²⁺ type detection as the index.

For example, by allowing a test compound to undergo the reaction for apredetermined period of time and using the reduction of intracellularCa²+concentration as the index, a substance having an IC₅₀ value of 10μM or less, preferably a substance having an IC₅₀ value of 1 μM or less,more preferably a substance having an IC₅₀ value of 0.1 μM or less canbe selected as an inverse agonist. By carrying out screening of inverseagonists by the Ca²⁺ type screening method of the present invention, asubstance which is useful as an agent for treating renal failure can bescreened.

2) cAMP Type Screening Method

The cAMP type screening method of the present invention is notparticularly limited, so far as it includes a step for detecting whetheror not it is an inverse agonist by the cAMP type detection method of thepresent invention and a step for selecting an inverse agonist.

The inverse agonist can be screened by the aforementioned cAMP typedetection method using increase of intracellular cAMP concentration ofthe cell for cAMP type detection as the index.

Using increase of intracellular cAMP concentration as the index, a testcompound having an ED₅₀ value of 10 μM or less (more preferably 1 μM orless) can be selected as a substance having inverse agonist activity.

By carrying out screening of inverse agonists by the cAMP⁺ typescreening method of the invention, a substance which is useful as anagent for treating renal failure can be screened.

3) GTPγS Binding Type Screening Method

The GTPγS binding type screening method of the present invention is notparticularly limited, so far as it includes a step for detecting whetheror not it is an inverse agonist by the GTPγS binding type detectionmethod of the present invention and a step for selecting an inverseagonist.

The inverse agonist can be screened by the aforementioned GTPγS bindingtype detection method, using reduction of specific GTPγS binding by atest compound as the index.

By carrying out screening of inverse agonists by the GTPγS binding typescreening method of the present invention, a substance useful as anagent for treating renal failure treating can be screened.

(2) Method for screening a substance which inhibits CTGF expression,using inhibition of activity of a polypeptide for screening (preferablyFGK) as the index

It is shown that a substance which inhibits expression of CTGF becomesan agent for treating renal failure. Accordingly, a substance which isuseful as an agent for treating renal failure can be screened by amethod of the present invention for screening a substance which inhibitsexpression of CTGF, namely, a method which has the feature comprising

a step of allowing a cell for screening expressing the CTGF promoterhaving a reporter gene in its downstream to contact with a testcompound, and

a step of measuring the reporter activity in the aforementioned cell.More illustratively, the aforementioned screening can be carried out bythe method described in Example 3 or Example 7. For example, by adding atest compound under the assay condition described in Example 3 orExample 7, a substance having an IC₅₀ value of 10 μM or less, preferablya substance having an IC₅₀ value of 1 μM or less, more preferably asubstance having an IC₅₀ value of 0.1 μM or less under the condition ofExample 3 or Example 7 can be selected as a substance which is useful asan agent for treating renal failure.

(3) Screening method which uses change in activity of a polypeptide forscreening (preferably FGK) as the index, using the COLIA2 promoter

A polypeptide for screening (preferably FGK) which activates thepromoter of CTGF as a target of an agent for treating renal failure isuseful by itself as a screening tool for an agent for treating a renalfailure. The inventors of the present invention have found that FGKactivates the promoter of COLIA2 as shown in Example 3 which isdescribed later, and a screening method which uses change in FGKactivity as the index using the COLIA2 promoter was established. As oneof the systems in which activity inhibition of activity of FGK isdetected in vitro, the following reporter assay system using the COLIA2promoter can be used.

The screening method of the present invention which uses change inactivity of a polypeptide for screening (preferably FGK) as the indexusing the COLIA2 promoter has the feature comprising

a step of allowing a cell for a screening tool expressing the COLIA2promoter having a reporter gene in its downstream to contact with a testcompound and

a step of measuring the reporter activity in the aforementioned cell.The screening method can be carried out by using the COLIA2 promoterinstead of the CTGF promoter in the aforementioned method for screeninga substance which inhibits CTGF expression. By carrying out theaforementioned steps and thereby selecting a substance which inhibitsthe reporter activity, a substance which is useful for treating renalfailure can be screened. More illustratively, the aforementionedscreening can be carried out by the method described in Example 3. Forexample, by adding a test compound under the assay condition describedin Example 3, a substance having an IC₅₀ value of 10 μM or less,preferably a substance having an IC₅₀ value of 1 μM or less, morepreferably a substance having an IC₅₀ value of 0.1 μM or less under thecondition of Example 3 can be selected as a substance which is useful asan agent for treating renal failure.

<Pharmaceutical Composition for Treating Renal Failure and ProductionMethod Thereof>

In the present invention, a pharmaceutical composition which comprisesan inverse agonist for the polypeptide for a screening tool [e.g., aDNA, a protein (includes an antibody or antibody fragment), a peptide orother compound] which can be selected, for example, by the screeningmethod of the present invention as an active ingredient is included.

Also, a method for producing a pharmaceutical composition for treatingrenal failure, which comprises (1) (i) a step of allowing a cell for ascreening tool or its cell membrane co-expressing a chimeric G proteinin which its C-terminal amino acid sequence is the amino acid sequencerepresented by SEQ ID NO:16, and which is a chimera of a partialpolypeptide having promoting activity of phospholipase C activity of aphospholipase C activity-promoting G protein with a partial polypeptidehaving a Gi receptor coupling activity, to contact with a test compound,and (ii) a step of analyzing a change in activity of a polypeptide for ascreening tool in the aforementioned cell, or (2) (i) a step of allowinga cell for a screening tool or its cell membrane expressing the DNA ofSEQ ID NO:13 or 14 having a reporter gene in its downstream to contactwith a test compound, and (ii) a step of analysis comprising measurementof the reporter activity in the aforementioned cell and then making theanalyzed substance into a pharmaceutical composition is included in thepresent invention.

In the present invention, a method for producing a pharmaceuticalcomposition for treating renal failure ahs the feature comprising a stepof carrying out screening using the screening method of the presentinvention and a step of preparing a pharmaceutical composition using asubstance obtained by the aforementioned screening is included.

As the active ingredient in the pharmaceutical composition of thepresent invention, an inverse agonist of the polypeptide for a screeningtool can be used, and the aforementioned inverse agonist can beselected, for example, by the screening method of the present invention.As the inverse agonist for the polypeptide for a screening tool, forexample, the compounds selected by the screening method of the presentinvention (cf. Examples 5 to 7 and Table 1 which are described later)can be cited. The pharmaceutical composition of the present invention isnot limited to a pharmaceutical composition which comprises a substanceobtained by the screening method of the present invention as the activeingredient. All of pharmaceutical compositions for treating renalfailure which comprise an inverse agonist for the polypeptide for ascreening tool as the active ingredient are included therein.

In this connection, confirmation of the presence of the effect oftreating renal failure can be carried out by a method commonly known tothose skilled in the art or a modified method thereof. The effect oftreating renal failure can be detected using inhibition oftubulointerstitial fibrosis, reduction of the amount of protein inurine, amount of blood creatinine or the like as the index. For example,with regard to the confirmation of the inhibitory effect ontubulointerstitial fibrosis, it can be confirmed using the UUO modelmouse described in the Non-patent Reference 15 or a non-patentreference: Kidney International, 2002, vol. 61, pp. 1684-1695, or amodified method thereof. More illustratively, it can be confirmed by thefollowing method. Male ICR mice are anesthetized by intraperitoneallyinjecting pentobarbital (50 mg/kg) (i.p.) at a liquid dose of 0.1 ml/10g. The UUO mice can be prepared by incising the left abdominal side,ligating the left urinary duct at two positions with 4-0 silk thread,cutting the space, and then stitching the incised part. The UUO mice aredivided into a control group and a test compound administration group,and an agent is administered by a method corresponding to its type.After the administration (e.g., 5 days after the preparation of UUO byonce a day administration), a blood sample is collected underpentobarbital anesthesia, and then left kidney (morbid state kidney) andright kidney (control kidney) are excised and their wet weights aremeasured. By dividing the kidney, a part thereof can be pathologicallyevaluated (e.g., Masson trichrome staining (collagen protein accumulatedin the interstitium is stained blue)). Also, by extracting RNA from apart thereof, expression of a fibrosis marker gene (e.g., CTGF,collagen, fibronectin or the like) can be detected by a conventionallyknown method (e.g., northern blotting or RT-PCR). When staining of thetest compound administration group is reduced (when collagen protein isreduced) in comparison with the control group by the aforementionedstaining, it can be judged that the test compound has the effect oftreating renal failure. Also, when expression quantity of a fibrosismarker in the test compound administration group is reduced incomparison with the control group by the aforementioned staining, it canbe judged that the test compound has the effect of treating renalfailure.

In addition, reduction of protein in urine can be confirmed, forexample, using the 5/6 kidney excision rat described in a non-patentreference Pharmacological Research, 2003, vol. 47, pp. 243-252 or amodified method thereof.

Among the confirming methods of the aforementioned effect of treatingrenal failure, most desirable one is the method which uses 5/6nephrectomized rat. More illustratively, it can be confirmed by thefollowing method. Wister rats of 9 weeks age are anesthetized byintraperitoneally injecting pentobarbital (50 mg/kg) (i.p.). A 5/6nephrectomized renal failure model rat can be prepared by incising theleft abdominal side, excising 2/3 of the left kidney, and then excisingwhole portion of the right kidney one week thereafter. The 5/6nephrectomized renal failure model rat are divided into a control groupand a test compound administration group, and an agent is administeredby a method corresponding to its type (e.g., once a day administrationstarting 2 weeks after the preparation of the 5/6 nephrectomized renalfailure model rat ). In every 1 week after the operation, measurement ofblood pressure and urine collection test in individual metabolism cageare carried out periodically (e.g., once a week for 10 weeks). At thetime of sacrifice, blood sample is collected from the abdominal venacava and kidney excision is carried out. The urine samples are used forthe measurement of the total volume, protein concentration and the like,and the blood samples are used for that of the concentration ofcholesterol, urea nitrogen, creatinine and the like, and the kidneytissue samples are used for the evaluation of pathological tissuechanges and the like. For example, when the amount of protein in urineis reduced, or when urea nitrogen quantity or creatinine quantity inblood is reduced in comparison with the control group, it can be judgedthat the test compound has the effect of treating renal failure.

The pharmaceutical preparation which comprises a substance or an inverseagonist of the polypeptide for a screening tool [e.g., a DNA, a protein(includes an antibody or antibody fragment), a peptide or othercompound], obtained by the screening method of the present invention asthe active ingredient can be prepared as a pharmaceutical compositionusing pharmaceutically acceptable carrier, excipient and/or otheradditives generally used for preparation, according to the type of theaforementioned active ingredient.

An administration includes such as oral administration through tablets,pills, capsules, granules, fine subtilaes, powders and oral solutions,and parenteral administration through injections such as intravenousinjections, intramuscular injections, intraarticular injections or thelike, suppositories, percutaneous administration preparations andtransmucosal administration preparations. Particularly in the case ofpeptides which are digested in the stomach, parenteral administrationsuch as intravenous injection is desirable.

In a solid composition for oral administration, one or more activesubstances can be mixed with at least one inert diluent such as lactose,mannitol, glucose, microcrystalline cellulose, hydroxypropylcellulose,starch, polyvinyl pyrrolidone and aluminum magnesium silicate. As usual,the aforementioned composition may contain other additives than theinert diluent, such as a lubricant, a disintegrating agent, astabilizing agent and a solubilizing or solubilization assisting agent.As occasion demands, tablets or pills may be coated with a sugar coatingor a film of a gastric or enteric substance.

The liquid composition for oral administration can include emulsions,solutions, suspensions, syrups, or elixirs and can contain a generallyused inert diluent such as purified water or ethanol. The aforementionedcomposition can contain additives other than the inert diluent such as amoistening agent, a suspending agent, a sweetener, an aromatic andantiseptic.

The injections for parenteral administration can include aseptic aqueousor non-aqueous solutions, suspensions or emulsions. As a diluent, theaqueous solutions or suspensions can include such as distilled water forinjection and physiological saline. Examples of a diluent of thenon-aqueous solutions and suspensions can include propylene glycol,polyethylene glycol, plant oil (e.g., olive oil), alcohols (e.g.,ethanol), polysorbate 80 and the like. The aforementioned compositioncan further contain a moistening agent, an emulsifying agent, adispersing agent, a stabilizing agent , a solubilizing or solubilizationassisting agent, an antiseptic or the like. The aforementionedcomposition can be sterilized by filtration through a bacteria retainingfilter, blending of a germicide or irradiation. Alternatively, a sterilesolid composition is produced, which can be used by dissolving insterile water or other sterile solvent for injection prior to its use.

The dose can be optionally decided by taking into consideration ofstrength of the activity of the active ingredient,-namely a substanceobtainable by the screening method of the invention, symptoms and age,sex and the like of each subject to be administered.

For example, in the case of oral administration, its dose is usuallyfrom about 0.1 to 100 mg, preferably from 0.1 to 50 mg, per day per anadult (body weight of 60 kg). In the case of parenteral administration,it is from 0.01 to 50 mg, preferably from 0.01 to 10 mg, per day, in theform of injections.

EXAMPLES

The present invention is described in detail in the following based onexamples, but the present invention is not limited by the examples. Inthis connection, unless otherwise noted, it is possible to carry out inaccordance with conventionally known methods (Maniatis, T. et al.,“Molecular Cloning—A Laboratory Manual”, Cold Spring Harbor Laboratory,NY, 1982, and the like).

Also, when commercially available reagents and kits are used, it ispossible to carry out in accordance with manuals attached to commercialproducts.

Example 1

Isolation of Gene Coding for FGK

A complete cDNA coding for the FGK of the present invention was obtainedby PCR using a human kidney cDNA (Clontech) as a template. Theoligonucleotide represented by SEQ ID NO:3 was used as the forwardprimer, and the oligonucleotide represented by SEQ ID NO:4 as thereverse primer (a XbaI site was added to each 5′ end). A DNA polymerase(Pyrobest DNA Polymerase; Takara Shuzo) was used in the PCR, and a cycleof 98° C. (30 seconds)/55° C. (30 seconds)/72° C. (2 minutes) wasrepeated for 30 cycles in the presence of 5% DMSO. As a result, a DNAfragment of about 1.0 kbp was amplified. This fragment was digested withXbaI and then cloned using pEF-BOS-dhfr plasmid (Mizushima, S. andNagata, S. (1990) Nucleic Acids Res., 18, 5322). The thus constructedplasmid was named pEF-BOS-dhfr-FGK. When nucleotide sequence of theobtained clone was analyzed by dideoxy terminator method using AB1377DNA Sequencer. (Applied Biosystems), the nucleotide sequence representedby SEQ ID NO:1 was obtained. The sequence has an open reading frame(ORF) of 993 bases. An amino acid sequence (330 amino acids) deducedfrom the ORF is shown in SEQ ID NO:2.

Example 2

Preparation of a Reporter Plasmid using Human Connective Tissue GrowthFactor (CTGF) Promoter and a Reporter Plasmid using Human Type ICollagen Alpha 2 Subunit (COLIA2) Promoter

A human genomic DNA derived from total blood (Human Genomic DNA;Clontech) was used as the template DNA for the amplification of promoterregions of human CTGF and human COLIA2 genes. For the amplification ofthe CTGF promoter region, an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO:5 was used as the forwardprimer, and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO:6 was used as the reverse primer. Also, for theamplification of the COLIA2 promoter region, an oligonucleotideconsisting of the nucleotide sequence represented by SEQ ID NO:7 wasused as the forward primer, and an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO:8 was used as the reverseprimer. In the PCR, a DNA polymerase (Pfu turbo DNA polymerase;Stratagene) was used, and after heating at 94° C. (2 minutes), a cycleof 94° C. (30 seconds)/58° C. (30 seconds)/72° C. (2 minutes) wasrepeated 35 times. As a result, a DNA fragment of about 1.2 kbp in thecase of the CTGF promoter, or of about 0.4 kb in the case of the COLIA2promoter, was amplified. Thereafter, the respective amplified productswere separated by 1% agarose gel and purified using a spin column(QIAquick Gel Extraction Kit; Qiagen), and then PCR was again carriedout using each of them as the template. For the amplification of theCTGF promoter region, an oligonucleotide consisting of the nucleotidesequence represented by SEQ ID NO:9 was used as the forward primer, andan oligonucleotide consisting of the nucleotide sequence represented bySEQ ID NO:10 was used as the reverse primer. Also, for the amplificationof the COLIA2 prombter region, an oligonucleotide consisting of thenucleotide sequence represented by SEQ ID NO:11 was used as the forwardprimer, and an oligonucleotide consisting of the nucleotide sequencerepresented by SEQ ID NO:12 was used as the reverse primer. In thiscase, a KpnI recognizing sequence was added to the 5′ end of the eachforward primer, and a BglII recognizing sequence was added to the 5′ endof each reverse primer. In the PCR, a DNA polymerase (Pfu turbo DNApolymerase; Stratagene) was used, and after heating at 94° C. (2minutes), a cycle of 94° C. (30 seconds)/58° C. (30 seconds)/72° C. (2minutes) was repeated for 30 times. Thereafter, the respective amplifiedproducts were separated by 1% agarose gel; purified using a spin column(QIAquick Gel Extraction Kit; Qiagen); digested with KpnI and BglII;inserted into a plasmid for reporter expression (Pickagene Basic Vector2; Toyo Ink); and then cloned. Nucleotide sequences of the obtainedclones were analyzed by dideoxy terminator method using AB13770 DNASequencer (Applied Biosystems), and a clone coincided with a known CTGFpromoter sequence (GenBank accession No. AL354866) or COLIA2 promotersequence (GenBank accession No. AF004877) was respectively selected. Thereporter plasmids obtained by the above respectively contained from−1,129 bp to +24 bp (SEQ ID NO:13) of the human CTGF promoter or from−337 bp to +22 bp (SEQ ID NO:14) of the human COLIA2 promoter, andrespectively named pCTGF-luc and pCOLIA2-luc.

Example 3

Construction of Reporter Assay System of CTGF Promoter and COLIA2Promoter and Effects by FGK Transfection

Using Dulbecco's modified Eagle's medium (DMEM) containing 10% fetalbovine serum, HEK293 cells (obtained from ATCC) were dispensed in 1×10⁵cells per well portions into a 24-well plate (Asahi Technoglass) andcultured at 37° C. for 24 hours. Transfection of 0.1 μg of a reporterplasmid and 0.5 μg at the maximum of a human FGK expression plasmidwhich is 0.6 μg in total per well was carried out using a transfectionreagent (FuGENE6; Boehringer-Roche) in accordance with the instructionsattached thereto. In this case, the amount of the transfected genes ineach of the all wells was adjusted to 0.6 μg in total per well by addinga plasmid vector (pcDNA3.1; Invitrogen). Cells transfected with thepcDNA3.1 vector alone without containing FGK, together with the reporterplasmid, was used as the control. The medium was discarded 24 hoursafter transfection, and the residue was lysed by adding 100 μl per wellof a cell lysis buffer. A 50 l portion of the lysate was mixed with 100μl of a luciferin substrate solution and allowed to undergo the reactionto be measured using a luminometer (ML3000; Dynatech Laboratories). Therelative activity to the luciferase activity of the control (control isdefined as 1) is shown in FIG. 1. Activation of the CTGF promoter andCOLIA2 promoter by the expression of FGK was observed. Additionslly,dose-dependent activation by FGK was also observed. Based on the above,it was revealed that FGK induces expression of CTGF and type I collagen.

By using the assay system, a substance which inhibits CTGF expressionand a substance which is useful as an agent for treating renal failurecan be screened.

Example 4

Construction of Screening System Which Uses FGK Activity Change as theIndex

An expression plasmid used in the Example for expressing a chimericprotein of Gq and Gi was prepared by cloning, a gene (hereinafter, to bereferred to as Gqi gene) constructed by replacing 5 amino acids in theGq C-terminal side (Glu-Tyr-Asn-Leu-Val; the amino acid sequencerepresented by SEQ ID NO:15) with 5 amino acids in the Gi C-terminalside (Asp-Cys-Gly-Leu-Phe; the amino acid sequence represented by SEQ IDNO:16) in accordance with the method of Conklin, B. R. et al. (Nature,363, 274-276, 1993) into the plasmid pEF-BOS-dhfr. The constructedplasmid was named pEF-BOS-Gqi.

As the illustrative method, firstly, using Dulbecco's modified Eagle'smedium (DMEM) containing 10% fetal bovine serum, HEK293 cells (obtainedfrom ATCC) were dispensed in 1×10⁵ cells per well portions into a24-well plate (Asahi Technbglass) and cultured for 24 hours. Thentransfection of 0.05 μg of a reporter plasmid pSRE-luc in which a serumresponse sequence was inserted into upstream of a luciferase gene(PathDetect™ SRE cis-Reporting System; Stratagene), 0.05 μg ofpEF-BOS-Gqi and 0.5 μg at the maximum of a human FGK expression plasmidwhich is 0.6 μg in total per well was carried out using a transfectionreagent (FuGENE6; Boehringer-Roche). Cells transfected with the pcDNA3.1vector alone without containing FGK, together with the reporter plasmidand pEF-BOS-Gqi, was used as the control. Other conditions andmeasurement of the reporter activity were as described in Example 3. Therelative activity to the luciferase activity of the control (control isdefined as 1) is shown in FIG. 2. Increase in the reporter activity bythe expression of FGK was observed. Additionally, dose-dependentactivation by FGK was also observed. Based on these results above,screening of a substance which inhibits FGK activity, namely a substancewhich is useful as an agent for treating renal failure by the assaysystem becomes possible.

Example 5

Screening of a Substance Which Inhibits FGK Activity

Screening of a test substance was carried out using the screening systemwhich uses changes in FGK activity as the index, constructed in Example4.

Using 10% fetal bovine serum-containing Dulbecco's modified Eag;e'smedium(DMEM),HEK293 cells(obtained from ATCC) were inoculated in 1×10⁴cells per well portions into a 96-well plate (Asahi Technoglass) andcultured at 37° C. for 24 hours. Transfection of 0.01 μg of the reporterplasmid pSRE-luc (Example 4), 0.03 μg of pEF-BOS-Gqi and 0.02 μg of ahuman FGK expression plasmid which is 0.06 μg in total per well wascarried out using a transfection reagent (FuGENE6; Boehringer-Roche).After 6 hours of incubation at 37° C., a dimethyl sulfoxide solution ofeach test compound was added to become a final concentration of 30 μM,10 μM, 3 μM, 1 μg, 0.3 μM or 0.1 μM, and then incubated at 37° C. foradditional 24 hours. Thereafter, the reporter activity was measured todetermine 50% activity inhibition concentration (IC₅₀) of each testcompound. As a control of the compound treatment, reporter activity whenthe same volume of dimethyl sulfoxide alone was added was regarded as100%. Other conditions and measurement of reporter activity were asdescribed in Example 4. Seven different compounds can be obtained when asubstance which inhibits the FGK activity in cells expressing FGK (asubstance having an IC₅₀ value of 10 μM or less) were selected asinverse agonists for FGK. Structures, activities and suppliers of thesecompounds are shown in Table 1.

Example 6

Detection of the Inhibition of CTGF Expression by a Substance WhichInhibits FGK Activity

Primary culture cells of human proximal renal tubule epithelium (RPTEC;Asahi Technoglass) were plated at 2×10⁵ cells per well into 12-wellplates (Asahi Technoglass) using a proliferation medium for kidneyepithelial cell use (Burette Kit REGM; Asahi Technoglass). Afterincubation for 24 hours at 37° C., each of the test compounds obtainedin Example 5 (substances which inhibit FGK activity, namely FGK inverseagonists) was added thereto to a final concentration of 10 μM, and thenincubated for additional 24 hours at 37° C. Thereafter, the cells werecollected and the total RNA was purified using a spin column (RNeasyMini Kit; Qiagen). Reverse transcription reaction was carried out using1.5 μg of the total RNA and a reverse transcriptase (PowerScriptTranscriptase; BD Biosciences) to obtain cDNA samples. Using each ofthem as the template, expressed amounts of CTGF and a housekeeping geneof glyceraldehyde 3-phosphate dehydrogenase (to be referred to as G3PDHhereinafter) were measured by real time PCR using PRISM 7700 SequenceDetector (PERKIN ELMER). In the PCR, SYBR Green PCR Master Mix (AppliedBiosystems) was used, and after heating at 50° C. (2 minutes) and 95° C.(10 minutes), a cycle of 95° C. (15 seconds)/60° C. (60 seconds) wasrepeated for 40 times. For the detection of CTGF, an oligonucleotiderepresented by SEQ ID NO:17 was used as the forward primer, and anoligonucleotide represented by SEQ ID NO:18 was used as the reverseprimer. For the detection of G3PDH, an oligonucleotide represented bySEQ ID NO:19 was used as the forward primer and an oligonucleotiderepresented by SEQ ID NO:20 was used as the reverse primer. A valuecalculated by dividing the expressed amount of CTGF by the expressedamount of G3PDH, both obtained by the PCR, was defined as the CTGFexpression quantity per unit RNA quantity. Also, as a control of thecompound treatment, the CTGF expression quantity per unit RNA quantitywhen the same volume of dimethyl sulfoxide alone was added was regardedas 100%. The inhibitory activity of CTGF expression obtained by theassay (inhibitory activity at a compound concentration of 10 μM) isshown in Table 1. It was able to confirm that the substances obtainedusing the inhibition of FGK activity as the index surely inhibit CTGFexpression.

Example 7

Screening of a Compound Which Inhibits CTGF Promoter Activation by FGK

Screening of a test compound constructed in Example 3 was carried outusing the CTGF promoter activation system by FGK.

HEK293 cells (obtained from ATCC) were plated at 1×10⁴ cells per wellinto 96-well plates (Asahi Technoglass) in Dulbecco's modified Eagle'smedium containing 10% fetal bovine serum (DMEM). After incubation for 24hours at 37° C., cells were transfected with 0.02 μg of pCTGF-luc (cf.Example 2) and 0.02 μg of a human FGK expression plasmid which is 0.04μg in total per well was carried out using a transfection reagent(FuGENE6; Boehringer-Roche). After incubation for 6 hours at 37° C., adimethyl sulfoxide solution of each test compound was added thereto tobecome a final concentration of 30 M, 10 μM, 3 μM, 1 μg, 0.3 μM or 0.1μg, and then incubated at 37° C. for additional 24 hours. Thereafter,the reporter activity was measured to determine 50% activity inhibitionconcentration (IC₅₀) of each test compound. As a control of the compoundtreatment, reporter activity when the same volume of dimethyl sulfoxidealone was added was regarded as 100%. Other conditions and measurementof reporter activity were as described in Example 3. Substances whichinhibit CTGF promoter activation by FGK were selected, and thesubstances having an IC₅₀ value of 10 μM or less were shown in Table 1.It was confirmed that a substance which inhibits CTGF expression by theassay system, namely a substance useful as a renal failure treatingagent can be certainly be screened. TABLE 1 Example 5 Inhibition Example7 of Example 6 Inhibition pGqi + pSRE- Inhibition of luc IntrinsicpCTGF- reporter CTGF mRNA luc Reference activity Expression activity NoSupplier code Compound name IC50 (μM) at 10 μM IC50 (μM) 1 ASINEX BAS1247186 N²-(4-ethoxyphenyl)- 6.1 62% 14.1 4′-methyl-4,5′-bi-1,3-thiazole-2,2′- diamine 2 ASINEX BAS 2936949 N-(5-chloro-2- 4.6 72%16.1 methoxyphenyl)-4-(2- thienyl)-1,3- thiazole-2-amine 3 ASINEX BAS0600431 N-[4′-methyl-2-[(3- 0.6 79% 2.6 methylphenyl)amino]-4,5′-bi-1,3-thiazol- 2′-yl]acetamide hydrobromate 4 MENAI LJ1100 LISTN-[4-(2-{[3- 3.9 65% 3.6 95 (trifluoromethyl) phenyl]amino}-1,3-thiazol-4- yl)phenyl]acetamide 5 ASINEX BAS 0600426 N-{2-[(4- 2.5 81%1.8 hydroxyphenyl)amino]- 4′-methyl-4,5′-bi- 1,3-thiazol-2′-yl}acetamide hydrobromate 6 MAYBRIDGE SEW 04180 2-[({4-[4- 6.8 35% 3.8(ethoxycarbonyl)- 3,5-dimethyl-1H- pyrazol-1- yl]phenyl}amino)carbonyl]benzoic acid 7 MERLIN MS 2357 N-{4-[2,5-dimethyl- 3.1 36% 1.73-(trifluoroacetyl)- 1H-pyrrol-1- yl]phenyl}-2- fluorobenzamide

Industrial Applicability

It was found that FGK as one of the a screening tools of the inventionactivates the promoter of CTGF. It is known that CTGF is a therapeutictarget for renal failure, by the use of the polypeptide as a screeningtool of the present invention and/or a cell expressing theaforementioned polypeptide, construction of a method for screening asubstance which inhibits CTGF expression using inhibition of theaforementioned polypeptide as the index, namely a method for screeningan agent for treating renal failure based on the inhibition of CTGFexpression by selecting an inhibitor of the aforementioned polypeptideand a method for screening an agent for treating renal failure byselecting an inverse agonist for the aforementioned polypeptide ispossible.

In addition, a pharmaceutical composition for treating renal failure canbe produced by using a substance as the active ingredient which isobtainable by the screening method of the present invention and makingit into a pharmaceutical composition using a carrier, an excipientand/or other additives.

Sequence Listing Free Text

An explanation of the “Artificial Sequence” is described in thenumerical entry <223> of the Sequence Listing. Illustratively,respective nucleotide sequences represented by the sequences of SEQ IDNOs:3, 4 and 9 to 12 are artificially synthesized primer sequences.

Although the invention has been described in the foregoing based on thespecific embodiments, various changes and modifications obvious to thoseskilled in the art are included within the scope of the presentinvention.

1. A screening tool for an agent for treating renal failure, which is apolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2, or a polypeptide comprising an amino acid sequence represented bySEQ ID NO:2 in which from 1 to 10 amino acids are deleted, substitutedand/or inserted and which is capable of activating CTGF promoter.
 2. Thescreening tool for an agent for treating renal failure, which is a cellexpressing the polypeptide described in claim
 1. 3. A method fordetecting whether or not a test compound is an inverse agonist, whichcomprises a step of allowing the cell described in claim 2 co-expressinga chimeric G protein in which C-terminal amino acid sequence is theamino acid sequence represented by SEQ ID NO:16, and which is a chimeraof a partial polypeptide having promoting activity of phospholipase Cactivity of a phospholipase C activity-promoting G protein with apartial polypeptide having a Gi receptor coupling activity, to contactwith a test compound, and a step of analyzing a change in activity ofthe polypeptide described in claim 1 in said cell.
 4. A method forscreening an agent for treating renal failure, which comprises a step ofallowing the cell described in claim 2 co-expressing a chimericG-protein in which C-terminal amino acid sequence is the amino acidsequence represented by SEQ ID NO:16, and which is a chimera of apartial polypeptide having promoting activity of phospholipase Cactivity of a phospholipase C activity-promoting G protein with apartial polypeptide having a Gi receptor coupling activity, to contactwith a test compound, and a step of analyzing a change in activity ofthe polypeptide described in claim 1 in said cell.
 5. A method forscreening a substance inhibiting expression of CTGF, which comprises astep of allowing the cell described in claim 2 expressing the DNA of SEQID NO:13 having a reporter gene in downstream to contact with a testcompound, and a step of measuring the reporter activity in said cell. 6.The screening method according to claim 5, wherein the substanceinhibiting expression of CTGF is an agent for treating renal failure. 7.A method for screening an agent for treating renal failure, whichcomprises a step of allowing the cell described in claim 2 expressingthe DNA of SEQ ID NO:14 having a reporter gene in downstream to contactwith a test compound, and a step of measuring the reporter activity insaid cell.
 8. A pharmaceutical composition for treating renal failure,which comprises an inverse agonist for the polypeptide described inclaim
 1. 9. A pharmaceutical composition for treating renal failure,which comprises a substance obtainable by the method according to one ofclaim 4 to claim
 7. 10. A method for producing a pharmaceuticalcomposition for treating renal failure, which comprises a step ofscreening using the method according to one of claim 4 to claim 7, and astep of preparing a pharmaceutical composition using a substanceobtained by said screening.
 11. A method for treating renal failure,which comprises administering an effective amount of an inverse agonistfor the polypeptide described in claim 1 and/or a substance obtaiable bythe method according to one of claim 4 to claim 7 to a subject in needof the treatment of renal failure.
 12. Use of an inverse agonist for thepolypeptide described in claim 1 and/or a substance obtainable by themethod according to one of claim 4 to claim 7 for the manufacture of apharmaceutical composition for treating renal failure.